Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 145 records · Page 8Linked to original sources

PubMed1995

Involvement of inflammatory cytokines in a delayed-type hypersensitivity reaction.

We previously reported the amino acid sequence of the human macrophage chemotactic factor with a molecular weight of 1000 (MCF-1) was WLGREDGSE. In this study we examined the roles of MCF-1 and tumor necrosis factor (TNF) in a guinea pig delayed-type hypersensitivity (DTH) reaction. When macrophage chemotactic activity (MCA) in a skin extract was subjected to gel filtration, only the activity with a molecular weight of 1000 was significantly inhibited by anti-human MCF-1 mAb (7-3 mAb). Both MCA and TNF activity were detected in skin extracts from DTH inflammation sites where the specific antigen was injected. Both activities were produced at the early DTH reaction stage. In contrast, MCF-1 was produced at the late DTH reaction stage. The erythema was suppressed significantly either by anti-guinea pig TNF Ab or by 7-3 mAb, suggesting the roles of these two cytokines in a DTH reaction.

Amino Acid Sequence↗

PubMed1995

Elevated promotion of prostacyclin production by synthetic lipid A analogs in aged human endothelial cells in culture.

We examined the effects of E. coli lipopolysaccharide (LPS) and synthetic analogs of lipid A, a bioactive moiety of LPS, on the prostacyclin (PGI2) production by young and old human endothelial cells in vitro. PGI2 production by endothelial cells has been shown to decrease during in vitro cellular senescence as well as in vivo. LPS and all the analogs tested in this study did not stimulate PGI2 production by young endothelial cells more than twofold. However, LPS and the majority of the lipid A analogs examined stimulated the PGI2 production by old cells more than twofold (approximately two- to sixfold). These results indicate that the responses to certain stimuli sometimes differ markedly between young and old cells, and this should be carefully considered when evaluating the biological effects of various compounds. Furthermore, these results suggest that certain synthetic lipid A analogs can be used as drugs to prevent some age-related vascular diseases.

Age Factors↗

PubMed1995

The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct.

Monoclonal antibodies directed against proteins modified with the major membrane lipid peroxidation product, 4-hydroxy-2-nonenal, have been established and characterized. The monoclonal antibodies specific for HNE-modified proteins were raised by immunizing mice with a HNE-keyhole limpet hemocyanin conjugate. The resulting five monoclonal antibodies (mAbs HNEJ-1-5) recognized HNE-modified bovine serum albumin (BSA), but not native BSA in Western blot studies. Of the five mAbs, HNEJ-2 exhibited the highest affinity for HNE-modified proteins and a much higher affinity for the HNE-histidine adduct than the HNE-lysine or HNE-cysteine adducts. mAb HNEJ-2 did not cross-react with proteins that had been treated with other aldehydes, such as 1-hexenal, 2-hexenal, 4-hydroxy-2-hexenal, 2-nonenal, formaldehyde, or glutaraldehyde. These results suggest that the major epitope recognized by mAb HNEJ-2 is the Michael addition-type HNE-histidine adduct.

Aldehydes↗

PubMed1995

cDNA cloning of mouse VLA-3 alpha subunit.

cDNA clones for mouse VLA (very late antigen)-3 alpha subunit (alpha 3 integrin) were isolated and sequenced. The encoded mouse alpha 3 integrin subunit was composed of 1,053 amino acid residues. The results of sequence analysis revealed similar structural characteristics of other VLA alpha subunits. For example, the presence of a large extracellular domain including three putative metal binding sequences, a transmembrane domain, and a short cytoplasmic domain. A higher level of its message was detected in thymus than in kidney, stomach, spleen, liver, brain, or lung by Northern blotting analysis.

3T3 Cells↗

PubMed1995

A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

Amino Acid Sequence↗

PubMed1995

Analysis of asparagine-linked oligosaccharides by sequential lectin affinity chromatography.

Lectins are proteins that specifically bind to a particular carbohydrate structure. Affinity chromatography with immobilized lectins is a quite effective technique not only for the fractionation of glycoproteins or oligosaccharides but also their structural assessment. In this article, we focus on the separation of glycopeptides and oligosaccharides derived from glycoproteins by affinity chromatography on immobilized lectin columns.

Agglutinins↗

PubMed1995

A high degree of sequence homology in the putative carbohydrate recognition domains of pokeweed mitogen and wheat germ agglutinin: poly-N-acetyllactosamine-binding lectins from different species.

The complete amino acid sequence of a poly-N-acetyllactosamine-binding pokeweed mitogen 4 (Pa-4) was determined using a protein sequencer. After digestion of Pa-4 with endoproteinase Lys-C, Asp-N, Arg-C or Glu-C, the resulting peptides were separated by reversed-phase high-performance liquid chromatography (HPLC) and then subjected to sequence analysis using a protein sequencer. The complete amino acid sequence of Pa-4 was found to exhibit a high degree of homology with that of wheat germ agglutinin (WGA) regarding their overall sequences and the spatial arrangement of cysteine-glycine. Furthermore, the amino acid residues of WGA directly involved in carbohydrate-binding sites were found in the homologous region in Pa-4. This is the first report to show that lectins from different plant families (Phytolaccaceae for Pa-4 and Gramineae for WGA) possess homologous primary sequences.

Amino Acid Sequence↗

PubMed1995

Protein kinase C phosphorylates p50 LSP1 and induces translocation of p50 LSP1 in T lymphocytes.

A lymphocyte-specific protein, p50, is phosphorylated on Ser and Thr residues in mitogen-activated T cells, suggesting that this molecule plays some role in the T cell activation cascade. p50 was identified as lymphocyte specific protein 1 (LSP1), which is a putative calcium-binding protein. In the present study, to clarify the role of p50 protein in the cascade, in vivo and in vitro phosphorylation of this molecule, and the effect of the phosphorylation on its distribution in activated T cells were examined. First, to obtain a sufficient amount of p50 as a phosphorylation substrate, p50 cDNA, which encodes a protein of 330 amino acid residues with a molecular mass of 36,728 Da, was cloned from an ICR mouse thymocyte cDNA library and expressed in Escherichia coli. When the putative coding region of p50 cDNA was expressed in E. coli, the product showed an apparent molecular mass of 50 kDa on SDS-PAGE. The recombinant p50 was phosphorylated in vitro by rabbit protein kinase C (PKC) and by murine cytosolic protein kinase, that was activated by a combination of phosphatidylserine and diacylglycerol. Furthermore, p50 was shown to be phosphorylated on the same sites in T cells upon stimulation with Con A as when phosphorylated in vitro by rabbit PKC, indicating that p50 is phosphorylated by PKC in Con A-stimulated T cells. On subcellular fractionation followed by immunoblotting analysis, membrane-bound p50 was shown to be released from the membrane following activation of PKC in T cells. These results and the recent finding that p50 binds to actin fibers raise the possibility that p50 controls the binding of actin fibers to the plasma membrane under regulation by PKC in T cells.

Amino Acid Sequence↗

PubMed1995

Fine structure of the epidermal basement membrane of the lip: applications of dithiothreitol separation and ultrathin frozen sectioning.

The fine structure of the epidermal basement membrane at the electron-microscopic level has already been defined. To obtain more details, two techniques, dithiothreitol separation and ultrathin frozen sectioning, were applied either alone or in combination. Negatively stained ultrathin sections showed a much thicker lamina densa than ordinary plastic-embedded sections. In the lamina lucida, bridging filaments and subbasal dense plates were observed in negative images. After the treatment with dithiothreitol, the lamina densa could be peeled off mechanically from the underlying dermis, and the anchoring fibrils were pulled off the dermis, preserving the connection with the lamina densa. With this specimen, bundles of anchoring fibrils were observed clearly and their lengths could be measured. Negatively stained ultrathin sections of dithiothreitol-separated specimens showed the fine structure of the lamina lucida. Bridging filaments in the lamina lucida were resolved by negative staining.

Animals↗

PubMed1995

Effects of various natural antioxidants on the Cu(2+)-mediated oxidative modification of low density lipoprotein.

We have reported in our previous paper that several flavan-3-ol derivatives (tea polyphenols) inhibited the Cu(2+)-mediated low density lipoprotein (LDL) oxidation in vitro. (-)-Epigallocatechin gallate (EGCG), in particular, exhibited strong inhibition. In this study, we have compared the antioxidative effects of EGCG with those of other natural antioxidants, such as flavonols, sesaminol, curcuminoid derivatives, tocopherol analogues and theaflavins. The antioxidative effects were monitored by conjugated diene formation in LDL which was carried out at 37 degrees C with 5 microM CuSO4 with or without antioxidants. Dibutyl hydroxytoluene (BHT) was used as a reference compound. The lag-time before the onset of conjugated diene formation was more than 100 min in the presence of 0.5 microM EGCG, theaflavin, myricetin, quercetin, and sesaminol. The ability to prolong the lag-time was in the order of sesaminol > quercetin > EGCG > theaflavin > or = myricetin > BHT > alpha-tocopherol. Among the 4 tocopherol analogues used, alpha-tocopherol showed the strongest antioxidative activity. We have also studied the effects of EGCG, BHT, and alpha-tocopherol on cholesteryl and alpha-tocopherol on cholesteryl ester (CE) degradation and apolipoprotein B 100 (apo B 100) fragmentation in the Cu(2+)-mediated oxidative modification of LDL. EGCG was the most effective inhibitor of CE degradation and apo B 100 fragmentation.

Antioxidants↗

PubMed1995

In vitro/in vivo evaluation of two series of TA-5707F controlled release matrix tablets prepared with hydroxypropyl methyl cellulose derivatives with entero-soluble or gel-formation properties.

Two series of matrix tablets for the newly developed anti-histaminergic drug, TA-5707F, were prepared: one series with an entero-soluble polymer, hydroxypropylmethylcellulose acetyl succinate (HPMC-AS), and one series with a hydrophilic gel-forming polymer, hydroxypropylmethylcellulose (HPMC). In the JP dissolution test of HPMC-AS tablets, as the soluble-pH of HPMC-AS was increased, the dissolution of the drug in the JP XII second fluid (pH 6.8) was increasingly delayed, and the duration time of the constant plasma level in fasted dogs was increased. However, the absorption of TA-5707F in fed dogs was markedly diminished. In the JP dissolution test, the HPMC tablets showed a zero order dissolution profile, being the same in both the JP first (pH 1.2) and the JP second fluids. The H-2 tablet, containing HPMC 60SH-4000 and lactose, showed a constant plasma level in fasted dogs. However, this tablet was disintegrated by the ingestion of food. The H-3 tablet containing a lot of HPMC 60SH-4000, but not lactose, showed insufficient duration in fasted dogs, but its plasma profile was less affected by food, and the propantheline bromide pretreatment extended the constant plasma level in dogs. Thus, the H-3 tablets were considered promising for human use. Then, H-2 and H-3 tablets were administered to humans; however, the plasma concentration profiles did not show sufficient duration time for either tablet. TA-5707F was considered to have been released slowly in human gastro-intestinal (GI) tract due to more moderate GI mobility.

Adult↗

PubMed1995

Preparation of controlled release tablets of TA-5707F with wax matrix type and their in vivo evaluation in beagle dogs.

Studies on controlled release dosage forms were conducted by using waxy materials for a newly developed anti-allergy drug, 6-methyl-N-(1H-tetrazol-5-yl)-2-pyridinecarboxamide (TA-5707F), which is not maintained at an effective level in blood for a long duration. Four kinds of tablets were prepared by changing the amount of hydrogenated oil (K3 wax) and polyethyleneglycol-6000 in the tablets. Then, they were administered orally to beagle dogs, and the TA-5707F concentration in the plasma was determined by a HPLC method. The pharmacokinetic parameters were estimated and compared with the results of the in vitro dissolution test determined by the JP paddle method and by the disintegration method. The linearity between the in vivo mean dissolution time (MDT) and in vitro MDT was good in both in vitro dissolution methods. However, the MDTs obtained by the disintegration method were one-third of the in vivo MDT, while those obtained by the paddle methods were 3 times higher. This suggests that both the diffusion of TA-5707F through the waxy matrix and the erosion of the wax matrix caused by the gastrointestinal (GI) tract mobility contributed to the in vivo dissolution mechanism. The blood levels were very low when the tablet was administered after giving food. The prolongation of resident time in the stomach and the low solubility of TA-5707F in an acidic medium seemed to be related to the phenomena. By the depression of GI motility using propantheline bromide, the blood levels could be markedly prolonged and the area under the plasma concentration-time curve (AUC) increased 1.3 times.

Animals↗

PubMed1995

Antioxidative activity of tetrahydrocurcuminoids.

In order to develop a new type of antioxidative compound which has both the phenolic and beta-diketone moiety in the same molecule, we converted three known curcuminoids, curcumin (diferuloylmethane, U1), (4-hydroxy-3-methoxycinnamoyl)methane (U2), and bis-(4-hydroxycinnamoyl)methane (U3), which are the natural antioxidants of Curcuma longa L. (tumeric), to tetrahydrocurcuminoids (THU1, THU2, and THU3, respectively) by hydrogenation, and evaluated their antioxidative activity by using linoleic acid as the substrate in an ethanol/water system. Further, we used the rabbit erythrocyte membrane ghost and rat liver microsome as in vitro systems and determined the antioxidative activity of these curcuminoids. When we evaluated their antioxidative activity by these assays, it was found that THU1 had the strongest antioxidative activity among all curcuminoids in each assay system. THU1 has been reported to be one of the main metabolites of U1 in vivo [Holder et al., Xenobiotica, 8, 761-768 (1978)]. These results suggest that THU1 must play an important role in the antioxidative mechanism of U1 in vivo by converting U1 into THU1.

Animals↗

PubMed1994

Interaction of immobilized recombinant mouse C-type macrophage lectin with glycopeptides and oligosaccharides.

Inflammatory and tumoricidal macrophages express galactose- and N-acetylgalactosamine-specific Ca(2+)-dependent lectins on their surfaces. This lectin is a family member of membrane-bound C-type animal lectins and consists of 304 amino acid residues (molecular weight 34,595). In the present study, expression vectors containing a nucleotide sequence corresponding to the carbohydrate-binding domain of mouse macrophage lectin cDNA have been prepared. The carbohydrate-binding specificity of the recombinant macrophage lectin expressed in Escherichia coli was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures on immobilized lectins. When elution profiles of high mannose-type and complex-type Asn-linked carbohydrate chains were compared, the degree of retardation from immobilized macrophage lectin column was in the order tetraantennary complex-type with terminal galactosyl residues > triantennary complex-type with terminal galactosyl residues > biantennary complex-type with terminal galactosyl residues > high mannose-type glycopeptides. N-Terminal octapeptides from human glycophorin A that bore three NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc serine/threonine-linked tetrasaccharide chains and their sequentially deglycosylated derivatives were also applied to this column. Glycopeptides carrying three constitutive GalNAc-Ser/Thr(Tn-antigen) had the strongest affinity, whereas those with fully sialylated carbohydrate tetrasaccharide chains showed weak interaction. The association kinetics of Asn-linked glycopeptides from bovine asialofetuin to recombinant macrophage lectin was determined by surface plasmon resonance spectroscopy. The results indicate k(assoc) value of 1.63 x 10(4) M-1 s-1. The calculated value for Ka was 6.20 x 10(7) M.

Amino Acid Sequence↗

PubMed1994

Strong affinity of Maackia amurensis hemagglutinin (MAH) for sialic acid-containing Ser/Thr-linked carbohydrate chains of N-terminal octapeptides from human glycophorin A.

The interaction of the Maackia amurensis hemagglutinin (MAH) with various glycopeptides and oligosaccharides was investigated by means of immobilized lectin affinity chromatography. An amino terminal octapeptide obtained from human glycophorin A having three Neu5Ac alpha 2-->3Gal beta 1-->3(Neu5Ac alpha 2-->6)GalNAc tetrasaccharide chains, designated as CB-II, was found to have an extremely strong affinity for MAH. Therefore, it is strongly suggested that hemagglutination by MAH was caused by its interaction with Ser/Thr-linked carbohydrate chains of human glycophorin A on erythrocyte membranes.

Carbohydrate Sequence↗

PubMed1994

Cloning and sequence analysis of the Maackia amurensis haemagglutinin cDNA.

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylated O-linked carbohydrate chains (Konami Y, Yamamoto K. Osawa T, Irimura T (1994) FEBS Lett 342:334-38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinated Maackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.

Amino Acid Sequence↗

PubMed1994

Sesaminol glucosides in sesame seeds.

The structures of novel sesaminol glucosides isolated from sesame seed were determined to be sesaminol 2'-O-beta-D-glucopyranoside, sesaminol 2'-O-beta-D-glucopyranosyl (1-->2)-O-beta-D-glucopyranoside and sesaminol 2'-O- beta-D-glucopyranosyl (1-->2)-O-[beta-D-glucopyransyl (1-->6)] -beta-D-glucopyranoside.

Carbohydrate Sequence↗

PubMed1994

Molecular cloning and characterization of prolyl endopeptidase from human T cells.

The prolyl endopeptidase (PEP) gene of human T cells was amplified by the PCR method and cloned in Escherichia coli. The complete gene consisted of 2,130 nucleotides corresponding to 710 amino acid residues with a calculated molecular mass of 80,750. The nucleotide sequence of this clone revealed that T cell PEP DNA is 48, 50, and 91% homologous to those of Flavobacterium meningosepticum, Aeromonas hydrophila, and porcine brain PEP, respectively. This gene was fused to the lacZ sequence from E. coli and expressed as a fused protein in E. coli. This fused protein exhibited PEP activity, which was inhibited by Z-Pro-prolinal, a specific inhibitor of PEP. The fused protein was purified on a beta-galactosidase specific affinity column. A polyclonal antibody was raised against the purified protein. Immunological characterization suggested that this protein is different from cytosol-soluble PEP.

Amino Acid Sequence↗